Deficiency of either vitamin B12 or folic acid can lead to an anemia characterized by megaloblastic hemopoiesis and a reduction in the number of red blood cells. The defect which causes the anemia is thought to be impaired DNA synthesis in marrow cells brought about by lack of functional folic acid coenzymes necessary for synthesis of DNA purines and thymidine. This proposal is designed to test this hypothesis by directly measuring the content of the folate coenzymes in bone marrow cells. This will be done on marrow cells from normal volunteers and from patients with vitamin B12 deficiency. These studies will also be done on marrow cells from rats made vitamin B12 deficient by feeding diets lacking the vitamin or by exposure to nitrous oxide. Alcoholism and anticonvulsant therapy have also been associated with folate deficiency. Therefore similar studies will be performed on marrow cells from rats fed alcohol containing liquid diets and from rats given dilantin in the drinking water. The content of folate coenzymes in liver, kidney, and brain of these rats will also be determined. Tissues will be extracted with hot solutions of sodium ascorbate to denature enzymes and protect labile reduced folates. The extracts will be treated with conjugase to hydrolyze polyglutamates and the individual folate monoglutamates will be separated by high-performance liquid chromatography (HPLC). Microbiological assays of HPLC eluate fractions with Lactobacillus casei will be used to quantitate each derivative. If these conditions lead to an altered distribution of folate derivatives in marrow cells and other tissues this will be taken as direct evidence of a functional deficiency of folic acid coenzymes similar to that proposed by the methyl trap hypothesis.